Polymer-based nanodiscs for studying structure and dynamics of G-protein-coupled receptors and other clinically relevant membrane proteins

整合膜蛋白需要膜模拟物在体外条件下保持可溶于水溶液。大多数情况下，这涉及在从生物膜溶解蛋白质以及随后的生物化学、生物物理或结构表征期间使用去污剂。不幸的是，许多与洗涤剂相关但众所周知不稳定的膜蛋白，如G蛋白偶联受体（GPCR），在洗涤剂胶束中是不稳定和无活性的。这干扰了生理相关的高分辨率结构的确定，并限制了我们对这些蛋白质内部工作的理解。最近，由苯乙烯和马来酸酐合成的共聚物已被证明在不需要洗涤剂的情况下溶解膜蛋白。在此过程中，聚合物通过包裹蛋白质及其天然脂质环境形成纳米大小的双层圆盘。在该提议中，我们使用这种聚合物技术用于未修饰的GPCR和其他与酶相关但不稳定的膜蛋白的无去污剂增溶、稳定和结晶。我们探索这种策略的潜力，从而避免广泛的蛋白质修饰，否则通常用于提高稳定性，但可能会带来难以预测的结构和功能的后果。作为原理证明，我们已经证明这些聚合物有效地从天然膜中溶解GPCR牛视紫红质，并且聚合物纳米盘为视紫红质提供比洗涤剂胶束更像膜的环境。在这里，我们研究了不同类型的聚合物形成稳定的和功能的nanodisces的能力，以及表征模型蛋白质，以及它们的适用性，在脂质立方相结晶。然后，我们扩展了该技术来分析更具挑战性的膜蛋白，这些膜蛋白在体外条件下在洗涤剂胶束中通常不稳定，迄今为止还没有进行彻底的功能和结构检查。为此，我们选择了GPCR人外周大麻素受体CB2和ABC转运蛋白人囊性纤维化跨膜传导调节因子（CFTR），它们分别参与多发性硬化和囊性纤维化的起源。结合生物化学和生物物理学的方法，包括，除其他外，X射线晶体学和电子顺磁共振光谱，我们推断这些关键蛋白质的生物物理和结构信息。这提高了我们对它们由配体结合、蛋白质相互作用和膜厚度或组成启动的信号传导机制的理解。膜蛋白天然状态结构的可用性对于发现具有更高选择性和亲和力的药物具有深远的意义。

Integral membrane proteins require membrane mimetics to remain soluble in aqueous solutions under in vitro conditions. Most often, this involves the use of detergents during both the solubilization of proteins from biological membranes as well as their subsequent biochemical, biophysical, or structural characterization. Unfortunately, many pharmacologically relevant but notoriously labile membrane proteins, such as G protein coupled receptors (GPCRs), are unstable and inactive in detergent micelles. This interferes with the determination of physiologically relevant high-resolution structures and limits our understanding of the inner workings of these proteins. Recently, a copolymer synthesized from styrene and maleic anhydride has been shown to solubilize membrane proteins without the need of detergents. In doing so, the polymer forms nanometer-sized bilayer discs by wrapping around the protein and its native lipid environment. In this proposal, we use this polymer technology for the detergent free solubilization, stabilization, and crystallization of unmodified GPCRs and other pharmacologically relevant but labile membrane proteins. We explore the potential of this strategy, thereby avoiding extensive protein modifications, which otherwise are often used to enhance stability but may entail poorly predictable structural and functional consequences. As a proof-of-principle, we have demonstrated that these polymers efficiently solubilize the GPCR bovine rhodopsin from native membranes and that the polymer nanodiscs provide a more membrane like environment for rhodopsin than detergent micelles do. Here, we examine the ability of different types of polymers to form stable and functional nanodiscs with well characterized model proteins as well as their suitability for crystallization in lipid cubic phases. Then, we expand the technique to analyze more challenging membrane proteins, which are typically unstable in detergent micelles under in vitro conditions and have thus far evaded a thorough functional and structural examination. To this end, we chose the GPCR human peripheral cannabinoid receptor CB2 and the ABC transporter human cystic fibrosis transmembrane conductance regulator (CFTR), which are involved in the origin of multiple sclerosis and cystic fibrosis, respectively. Following a combined biochemical and biophysical approach including, among others, X ray crystallography and electron paramagnetic resonance spectroscopy, we deduce biophysical and structural information on these crucial proteins. This improves our understanding of their signaling mechanisms initiated by ligand binding, protein protein interactions, and membrane thickness or composition. The availability of native-state structures of membrane proteins will have far reaching implications for the discovery of drugs with higher selectivity and affinity.